Flow method and apparatus for screening chemicals using micro x-ray fluorescence

ABSTRACT

Method and apparatus for screening chemicals using micro x-ray fluorescence. A method for screening a mixture of potential pharmaceutical chemicals for binding to at least one target binder involves flow separating a solution of chemicals and target binders into separated components, exposing them to an x-ray excitation beam, detecting x-ray fluorescence signals from the components, and determining from the signals whether or not a binding event between a chemical and target binder has occurred.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 11/444,660 filed May 31, 2006, which is a continuation-in-partof U.S. patent application Ser. No. 11/125,036 filed May 9, 2005, nowabandoned, which is a continuation of U.S. patent application Ser. No.10/206,524 filed Jul. 25, 2002, now abandoned, all hereby incorporatedby reference.

STATEMENT REGARDING FEDERAL RIGHTS

This invention was made with government support under contract numberW-7405-ENG- 36 and its successor contract number DE-AC52-06NA25396awarded by the U.S. Department of Energy. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to detecting binding events andmore particularly to a flow method for detecting binding events betweena potential pharmaceutical chemical and a target binder usingmicro-x-ray fluorescence spectroscopy.

Pharmaceutical chemicals are the active ingredients in drugs such as thenow popular Prilosec™, Lipitor™, Zocor™, Prozac™, Zoloft™, andCelebrex™, and it is believed that their pharmaceutical properties arelinked to their ability to bind to the “binding site” of one or moreproteins. The binding properties of a protein largely depend on theexposed surface amino acid residues of the polypeptide chain (see, forexample, Bruce Alberts et al., “Molecular Biology of the Cell’, 2ndedition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al.,“Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000).These amino acid residues can form weak noncovalent bonds with ions andother molecules. Effective binding generally requires the formation ofmany weak bonds at the “binding site” of the protein. The binding siteis usually a cavity in the protein formed by a specific arrangement ofamino acids. There must be a precise fit with the binding site foreffective binding to occur. The shapes of binding sites may differgreatly among different proteins, and even among different conformationsof the same protein. Even slightly different conformations of the sameprotein may differ greatly in their binding abilities. For thesereasons, it is extremely difficult to predict which chemicals will bindeffectively to proteins.

It can take many years to identify an effective pharmaceutical chemical.The desire to hasten the identification of important pharmaceuticalchemicals is a constant challenge that has prompted the use or screeningstrategies for screening a large number of structurally or chemicallyrelated materials, known in the art as a “library”, for bindingproperties to proteins.

Screening methods generally involve combining potential pharmaceuticalchemicals with target binders and determining which, if any, of thepotential pharmaceutical chemicals bind to any of the target binders.Potential pharmaceutical chemicals are preferably water-soluble organiccompounds that can dissolve into the blood stream. Target binders aregenerally biological materials such as enzymes, non-enzyme proteins,DNA, RNA, microorganisms (e.g. prions, viruses, bacteria, and the like),human cells, plant cells, animal cells, and the like. Potentialpharmaceutical chemicals that bind to at least one target binder arelikely candidates for further investigation of pharmaceutical properties(e.g. efficacy and toxicity).

Some of the known screening methods are described in the following threepatents.

U.S. Pat. No. 6,147,344 to D. Allen Annis et al. entitled “Method forIdentifying Compounds in a Chemical Mixture”, which issued Nov. 14,2000, describe a method for automatically analyzing mass spectrographicdata from mixtures of chemical compounds.

U.S. Pat. No. 6,344,334 to Jonathan A. Ellman et al. entitled“Pharmacophore Recombination for the Identification of Small MoleculeDrug Lead Compounds”, which issued Feb. 5, 2002, describes a method foridentifying a drug lead compound that inhibits binding of targetbiological molecules by contacting these target biological moleculeswith a library of cross-linked, target, binding fragments.

U.S. Pat. No. 6,395,169 to Ole Hindsgaul et al. entitled “Apparatus forScreening Compound Libraries”, which issued May 28, 2002, describes anapparatus that employs frontal chromatography combined with massspectometry to identify and rank members of a library that bind totarget receptor.

Screening methods sometimes employ tagged materials because theanalogous untagged material is otherwise not visible using theanalytical technique chosen for the screening method. Tagging mayinvolve attaching a labeled chemical portion to a chemical. An exampleof a screening method requiring tags is fluorescence activated cellsorting. An example of this method involves preparing a solution ofcells and antibodies bearing a fluorescent tag. Some of the antibodiesbind to some of the cells. One at a time, the cells flow past a laserbeam and a detector (such as a ultraviolet/visible fluorescencedetector). Cells that fluoresce (are bound to the tagged antibodies) andare then deflected into a collector (see, for example, Bruce Alberts etal., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing,Inc., New York, 1989, pages 159-160).

It is generally assumed that the attachment of a fluorescent tag onlyserves to make visible the otherwise invisible chemical and/or targetbinder, and does not alter the binding properties of the untaggedanalog. Since it is well known that even small changes to the structureof a chemical or target binder may affect its function, this assumptionmay not be a valid one. Tagged surrogates are structurally differentfrom their untagged counterparts, and these structural differences couldaffect their binding properties.

An efficient method for screening potential pharmaceutical chemicals forbinding to target binders remains highly desirable.

SUMMARY OF THE INVENTION

In accordance with the objects and purposes of the present invention, asembodied and broadly described herein, the present invention includes amethod for determining whether or not chemical binding occurs between atleast one target binder and at least one chemical to form a boundcomplex. The method includes preparing a solution comprising at leastone chemical and at least one target binder; flow-separating thesolution into at least two separated components; exciting atoms of thechemical and of a chemical portion of any bound complex present in anyflow separated component with a polychromatic x-ray excitation beam inorder to produce an x-ray fluorescence signal therefrom; detecting thex-ray fluorescence signal produced from the excited atoms present in thechemical and the chemical portion of any bound complex present in aseparated component; and determining from the x-ray fluorescence signalproduced from the excited atoms present in the chemical and the chemicalportion of any bound complex present in a separated component whether ornot any separated component comprises a bound complex.

The invention also includes a method for determining whether or notchemical binding occurs between at least one target binder and at leastone chemical to form a bound complex. The method includes preparing asolution comprising at least one chemical and at least one targetbinder; flow separating the solution into at least two separatedcomponents; exciting atoms of the chemical and of a chemical portion ofany bound complex present in any flow-separated component using an x-rayexcitation beam comprising x-rays of less than 9 KeV in order to producean x-ray fluorescence signal therefrom; detecting the x-ray fluorescencesignal produced from the excited atoms present in the chemical and inthe chemical portion of any bound complex present in a separatedcomponent; and determining from the x-ray fluorescence signal producedfrom the excited atoms present in the chemical and the chemical portionof any bound complex present in a separated component whether or not anyseparated component comprises a bound complex.

The invention also includes method for determining whether or notchemical binding occurs between at least one target binder and at leastone chemical to form a bound complex. The method involves preparing asolution comprising at least one chemical and at least one targetbinder; flow-separating the solution using a pressure gradient into atleast two separated components; exciting atoms of a chemical and of achemical portion of any bound complex present in any flow-separatedcomponent using an x-ray excitation beam to produce an x-rayfluorescence signal therefrom; detecting the x-ray fluorescence signalproduced from the excited atoms present in the chemical and the chemicalportion of any bound complex present in a separated component; anddetermining from the x-ray fluorescence signal produced from the excitedatoms present in the chemical portion of any bound complex present in aseparated component whether or not any separated component comprises abound complex.

The invention also includes an apparatus for screening chemical binding.The apparatus includes a flow separator for separating a solution ofchemicals and at least one target binder into at least twoflow-separated components; an x-ray excitation source for exposing atleast one of said flow-separated components to an x-ray excitation beamto produce an x-ray fluorescence signal therefrom; an x-ray detector fordetecting the x-ray fluorescence signal emitted from a flow-separatedcomponent; and a pump engaged with said flow separator for providing apressure gradient along said flow separator.

The invention also includes an apparatus for screening chemical binding.The apparatus includes a flow separator for separating a solution intoat least two flow separated components, the solution comprising at leastone chemical and at least one target binder; a polychromatic x-rayexcitation source for exposing at least one of the flow-separatedcomponents to an x-ray excitation beam to produce an x-ray fluorescencesignal therefrom; and an x-ray detector for detecting the x-rayfluorescence signal emitted from a flow-separated component.

The invention also includes an apparatus for screening chemical binding.The apparatus includes a flow separator for separating a solution ofchemicals and at least one target binder into at least twoflow-separated components; an x-ray excitation source for exposing atleast one of said flow-separated components to an x-ray excitation beamcomprising x-rays of less than 9 KeV to produce an x-ray fluorescencesignal therefrom; and an x-ray detector for detecting the x-rayfluorescence signal emitted from a flow-separated component.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiment(s) of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIGS. 1 a-b show typical process flow diagrams for the invention;

FIG. 2 shows a spectrum of polychromatic light produced using a Rhodiumtarget excitation source.

FIG. 3 shows a schematic representation of an embodiment apparatus ofthe invention; and

FIG. 4 shows an embodiment separator/sorter for sorting flow-separatedcomponents of a solution.

DETAILED DESCRIPTION OF THE INVENTION

Briefly, the present invention includes a method for identifying bindingevents between potential pharmaceutical chemicals and target binders.The method involves modifying a mixture of potential pharmaceuticalchemicals by adding at least one target binder to the mixture. Afterallowing sufficient time for any bound complex between any of thepotential pharmaceutical chemicals and any of the target binders toform, if such a complex can form, the resulting solution is flowseparated into at least two components. Each component is exposed to anx-ray excitation beam. If the exposed component emits a detectable x-rayfluorescence signal, that component is isolated. The identity of anyisolated component can be determined using one or more standardanalytical techniques, such as gas chromatography, liquidchromatography, mass spectrometry, nuclear magnetic resonancespectroscopy, infrared spectroscopy, ultraviolet spectroscopy, visiblespectroscopy, elemental analysis, cell culturing, immunoassaying, andthe like.

The method of the invention uses x-ray fluorescence as a probe to detectbinding events. X-ray fluorescence is a powerful technique that has beenused to determine the chemical elements that are present in a chemicalsample, and to determine the quantity of those elements in the sample.The underlying physical principal of the method is that when an atom ofa particular element is irradiated with x-ray radiation, the atom ejectsa core electron such as a K shell electron. The resulting atom is in anexcited state, and it can return to the ground state by replacing theejected electron with an electron from a higher energy orbital. This isaccompanied by the emission of a photon, i.e. x-ray fluorescence, andthe photon energy is equal to the difference in the energies of the twoelectrons. Each element has a characteristic set of orbital energies andtherefore, a characteristic x-ray fluorescence spectrum.

Many popular pharmaceutical chemicals, such as Prilosec™, Lipitor™,Zocor™, Prozac™, Zoloft™, and Celebrex™, contain the elements fluorine,chlorine, and/or sulfur. X-ray fluorescence is especially suited fordetecting potential pharmaceutical chemicals because it can be used todetect and quantify these elements, and in general, to detect andquantify any element with an atomic number of nine or higher.

The invention also includes an apparatus for screening a mixture ofpotential pharmaceutical chemicals for binding to at least one targetbinder. The apparatus includes a container for containing a solution ofa mixture of chemicals and at least one target binder. The apparatusalso includes a flow separator for separating the solution into at leasttwo separated components. The apparatus also includes an x-rayexcitation source for exposing at least one of the flow-separatedcomponents to an x-ray excitation beam. The apparatus also includes anx-ray detector for detecting an x-ray fluorescent signal emitted from aflow-separated component, a diverter for diverting a chosenflow-separated component, and a container for isolating the chosen,flow-separated component.

An x-ray fluorescence spectrometer includes an x-ray excitation sourceand an x-ray detector. It is capable of irradiating a sample with anx-ray beam, detecting the x-ray fluorescence from the sample, and usingthe x-ray fluorescence to determine which elements are present in thesample and providing the quantity of these elements. The x-rayfluorescence spectrometer used to demonstrate the invention was thecommercially available EDAX Eagle XPL energy dispersive x-rayfluorescence spectrometer, equipped with a microfocus x-ray tube,lithium drifted silicon solid-state detector, processing electronics,and vendor supplied operating software.

The use of capillary electrophoresis with x-ray fluorescence has beendescribed by Mann et al. in “Element Specific Detection in CapillaryElectrophoresis Using X-Ray Fluorescence Spectroscopy”, AnalyticalChemistry, vol. 72, pp. 1754-1758, (2000), incorporated by referenceherein. Mann et al. report the preparation of a mixture of chelationcomplexes of CDTA (cyclohexane diamine tetraacetic acid) and subsequentseparation using capillary electrophoresis. The separated complexes weredetected using a synchrotron-generated monochromatic, 10 keV x-ray beam.

The practice of the invention can be further understood with theaccompanying figures. Similar or identical structure is identified usingidentical callouts. FIGS. 1 a-b show typical process flow diagrams forthe invention. According to the invention, potential pharmaceuticalchemicals from optional reservoir 12 are combined with at least onetarget binder from target binder reservoir 14 to form a solution inreservoir 16. Potential pharmaceutical chemicals used with the inventionare typically water soluble organic chemicals, and have at least oneelement with an atomic number of nine or greater. Preferably, theyinclude at least one element selected from fluorine, chlorine, bromine,iodine, sulfur, phosphorus, selenium, lanthanum, cerium, praseodymium,neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium,erbium, thulium, ytterbium, lutetium, antimony, bismuth, and arsenic.Target binders that can be used with the invention include enzymes,non-enzyme proteins, DNA, RNA, plant cells, animal cells, human cells,and microorganisms (e.g. comprise prions, viruses, bacteria) and thelike.

The solution of the mixture of potential pharmaceutical chemicals andtarget binder(s) enters flow separator 18 (see FIG. 1 a), or firstenters pump 17 and then flow separator 18 (see FIG. 1 b). Flow separator18 uses a mobile phase to flow separate the solution into at least twocomponents. The target binder and chemical may be introduced into theflow separator at different times and/or at different locations alongthe flow separator. Flow separators that can be used with the inventioninclude, but are not limited to, centrifuges, cell sorters, orchromatographs (e.g. liquid chromatograph such as high performanceliquid chromatographs and fast protein listed chromatographs;electrophoretic separators such as capillary electrophoretic separators,gel filtration chromatographs, gel permeation chromatographs, sizeexclusion filters, dialysis filters, and the like). Preferably, theseparator is a capillary electrophoresis separator, i.e. a long thintube with a mobile phase (e.g. an aqueous buffer solution) inside thetube, and an electric potential across the length of the tube.

As the mixture separates into components, they are exposed to x-rays.After x-ray excitation source 20, preferably a rhodium target x-raytube, delivers a polychromatic x-ray beam 22 to a separated component,that component may or may not emit an x-ray fluorescence signal 24,which is detected by x-ray fluorescence detector 26, x-ray detectorsthat can be used with the invention include, but at not limited to,lithium-drifted silicon detectors, silicon drift detectors, or PINdiodes. If the exposed component does not emit an x-ray fluorescencesignal, that component is directed to first collector 28. If the exposedcomponent emits a fluorescence signal that is detected by x-rayfluorescence detector 26, it is directed to second collector 30. Thiscomponent is expected to include at least one bound complex of potentialpharmaceutical chemical and target binder.

A polychromatic x-ray excitation source (a rhodium source, for example)useful with the invention preferably provides x-rays having energiesless than about 9 KeV, and more preferably less than about 8 KeV. Thepolychromatic source preferably also provides x-rays having energiesgreater than about 12 KeV. FIG. 2 shows an energy spectrum of anexemplary Rhodium source, which provides polychromatic x-rays havingenergies less than 9 KeV and greater than 12 KeV. It should beunderstood that other target x-ray excitation sources besides Rh couldalso be used.

While only a first collector and a second collector are shown in FIGS. 1a-b, it should be understood that more collectors may be used, dependingon the number of separated components that are isolated from themixture. In another embodiment of the invention, no collectors are used.

The separated component that emits a detectable x-ray fluorescencesignal, i.e. the component directed to second collector 30, may then besent to analyzer 32. Analyzers that can be used with the inventioninclude, but are not limited to, gas chromatographs, liquidchromatographs, mass spectrometers, nuclear magnetic resonancespectrometers, infrared spectrometers, ultraviolet-visible (UV-VIS)spectrometers, fluorimeters, combustion analyzers (for elementalanalysis), cell cultures, immunoassays, and the like. The choice ofanalyzer will depend on the nature of the potential pharmaceuticalchemicals and/or binders being analyzed.

FIG. 3 shows a schematic view of an embodiment of a screening apparatusof the invention. As FIG. 3 shows, screening apparatus 34 includes inletmobile phase reservoir 36, which provides the mobile phase 38 forcapillary separator 40. Inlet end 42 of separator resides in inletmobile phase reservoir 36, while outlet and 44 resides in outlet mobilephase reservoir 46. After mobile phase 38 fills separator 40, an amountof a mixture of potential pharmaceutical chemicals and at least onetarget binder is introduced into inlet end 42 of separator 40. Inlet end42 is then replaced into mobile phase reservoir 36. An electricpotential between inlet end 42 and outlet end 44 of separator 40 drivesthe flow of the mobile phase 38 and of the mixture through separator 40.As FIG. 3 shows, component 48 has separated from the mixture. FIG. 3also shows x-ray excitation source 20 directing x-ray excitation beam 22at separated component 48, which then emits x-ray fluorescence signal 24that is detected by x-ray fluorescence detector 26. The detection of anemitted x-ray fluorescence signal triggers diversion valve 50, whichdiverts the flow of mobile phase 36 and separated component 48 todiverter 52, which directs mobile phase 36 and separated component 48 tocomponent collector 54.

The separation previously described was achieved using an electricpotential, which provided an electric gradient across the length ofcapillary separator 40. The separation can also be achieved by applyinga pressure gradient along the length of the tub. In this embodiment, thetube would include a stationary phase; a sample injection inlet would beused to introduce the solution into the tube, and a pump would providethe pressure gradient, as it does for high performance liquidchromatography.

As FIG. 3 shows component 48 is separated along a horizontal portion ofcapillary separator 40. This particular configuration is likely notoptimal for separating complexes derived from using microorganism orcell target binders. For these target binders, a separator/sorter thatseparates along a vertical portion is preferred.

FIG. 4 shows an embodiment of such a separator/sorter that can be usedwith the invention. Separator/sorter 56 can be used for separating andsorting mixtures derived from cells, microorganisms, microspheres havingattached proteins or nucleic acids, and the like. Separator/sorter 56includes vertical separator 58 through which separation occurs. As FIG.4 shows, the mixture has been separated into component 48 and component60. Component 48 is had been subjected to x-ray beam 22 from x-rayexcitation source 20 has emitted an x-ray fluorescence signal, which wasdetected by x-ray fluorescence detector 26. This triggered a response inapplied voltage course 62, which applies a voltage that deflectscomponent 48 into collector 64. If component 60 does not emit adetectable x-ray fluorescence signal, no voltage will be applied todeflect component 60 and it will flow into collector 66. However, ifcomponent 60 emits a detectable x-ray fluorescence signal, a voltagewill be applied to deflect component 60 and it will flow into collector68.

Separator/sorter 56 may include a laser source and associated detectorsfor performing conventional fluorescence activated cell sorting of thetype described by Bruce Alberts et al., “Molecular Biology of the Cell”,2nd edition, Garland Publishing, Inc., New York, 1989, pages 159-160.

If a pharmaceutical chemical is needed to bond to a specific targetbinder protein, for example, a large number of different potentialpharmaceutical chemicals can be screened according to the invention forbinding to that protein. The invention can be used to distinguish whichof the potential pharmaceutical chemicals bind strongly to the proteinfrom those that bind weakly or not at all. The protein would be combinedwith about 10 to 10,000 potential pharmaceutical chemicals, wherein eachof the potential pharmaceutical chemicals includes at least one elementhaving an atomic number of nine or higher. Preferably, the potentialpharmaceutical chemicals include an element having an atomic number ofnine or higher that is not found in the target binder to simplify thescreening method.

The invention could be used to, for example, determine whether eithercobalt ion (Co²⁺) and/or cyanocobalamin bind to the known, biologicallyactive protein Ure2p (see Kuruvilla et al., “Dissecting GlucoseSignaling With Diversity-Oriented Synthesis and Small-MoleculeMicroarrays”, Nature, Vol. 416, pp. 653-657). An aqueous solution ofcobalt (II) nitrate and cyanocobalamin would be added to Ure2p. Theresulting aqueous solution would be flow separated according to theinvention using, for example, a capillary electrophoresis separator. Anycomplex formed between the Ure2p and Co²⁺ and/or cyanocobalamin shouldhave a retention time that differs from either Co²⁺ or cyanocobalamin,would emit a detectable x-ray fluorescence signal, and would be isolableusing the invention.

The separation could be performed using, for example, a fused silicacapillary tube (POLYMICRO TECHNOLOGIES™) having the followingdimensions: 70 cm in length, 100 μm outer diameter (od), and a BERTAN™Model ARB-30 high voltage power supply to provide the electricpotential. The tube could be conditioned by first flushing it with a 1.0molar (M) solution of NaOH for 15 min, then rinsing with distilled,de-ionized water for 15 min, and then flushing and filling with 75 mMTrisma run buffer (pH 8.0) for an additional 15 min.

A baseline was obtained by introducing an aqueous mixture of cobaltnitrate (Co(NO₃)₂, and cyanocobalamin (10.2 mM) into the capillary tube,applying a potential of 10 kV between the ends of the tube, andseparating the mixture into its components. An EDAX™ Eagle II microx-ray fluorescence system equipped with a Rh target excitation sourceand a SiLi detector was used to interrogate each separated component andmeasure any emitted x-ray fluorescence signal. The x-ray tube of thesystem was operated at 40 kV and 1000 μ. The CoK_(α) x-ray emission wasmonitored to detect unbound Co²⁺ and cyanocobalamin. The spectrumacquisition time was about 10 seconds (s). The peak due to unbound Co²⁺was detected at about 4.5 min with a full-width-at-half-maximum (FWHM)of about 1 min. The cyanocobalamin peak was detected at about 8.5 minwith a FWHM of about 1.5 min.

Similarly, the invention could be used to determine whether ferritinand/or cyanocobalamin bind to Ure2. An aqueous solution of ferritin andcyanocobalamin would be added to Ure2. The resulting aqueous solutioncould be flow separated using a capillary electrophoresis separator.When exposed to an x-ray beam, the iron in ferritin and the cobalt incyanocobalamin each emit distinct and detectable x-ray fluorescencesignals that could be used to determine whether a complex betweenferritin and/or cyanocobalamin and Ure2p is formed.

A baseline was obtained as follows: A capillary electrophoresisseparator was prepared using a Bertan™ Model ARB-30 high voltage powersupply to provide the separation potential and a fused silica capillarytube (Polymicro Technologies™) having the following dimensions: 70 cm inlength, 100 μm inner diameter (id), 170 μm outer diameter (od). The tubewas conditioned by first flushing it with a 1.0 molar (M) solution ofNaOH for 15 min, then rinsing with distilled, de-ionized water for 15min, and then flushing with 100 mM Trisma run buffer (pH 8.0) for anadditional 15 min.

An aqueous solution of ferritin (1.16 mg/ml) and cobalamin (10.2 mM) wasintroduced into the capillary tube. After a separation potential of 9.5kV was applied between the ends of the tube, the solution flowed throughthe tube and separated into two components. An EDAX™ Eagle II microx-ray fluorescence system equipped with a Rh target excitation sourceand a SiLi detector was used to interrogate each separated component andmeasure any emitted x-ray fluorescence signal. The x-ray tube of thesystem was operated at 40 kV and 1000 μA. The CoK_(α) and FeK_(α) x-rayemission lines were monitored to detect the Fe³⁺ bound ferritin andcobalamin. The spectrum acquisition time was about 10 seconds (s). Thepeak due to Fe³⁺ of ferritin was detected at about 9.3 min with afull-width-at-half-maximum (FWHM) of about 1.7 min. The cyanocobalaminpeak was detected at about 6.3 min with FWHM of about 1 min.

The invention can be used in pharmaceutical metabolite studies to detectdangerous metabolic byproducts of a potential pharmaceutical chemical. Apotential pharmaceutical chemical having at least one atom with anatomic number of nine or higher could be given to a rat (or other testanimal). A blood sample would be taken from the rat before administeringthe potential pharmaceutical chemical to provide a baseline. Afteradministering the potential pharmaceutical, blood from the rat would beexamined for the presence of metabolites using the method of theinvention.

In summary, the present invention provides an apparatus and method fordetecting binding events between potential pharmaceutical chemicals andtarget binders. The present invention uses micro-x-ray fluorescence todetermine the presence and relative amounts of elements such asfluorine, chlorine, bromine, iodine, phosphorus, and sulfur, the lattertwo being important constituents of enzymes, non-enzyme proteins, DNA,and RNA. Thus, the invention provides a non-destructive method ofscreening the binding of potential pharmaceutical chemicals with atarget binder such as a protein or a nucleic acid. Known methods oftenrequire that the binder and/or potential pharmaceutical chemical includea covalently bound tag that fluoresces upon exposure to ultravioletexcitation radiation. By contrast, the invention does not require taggedmaterials.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practice application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

1. An apparatus for screening chemical binding, comprising: a flowseparator configured to separate a solution of chemicals and at leastone target binder into at least two flow-separated components; an x-rayexcitation source configured to expose at least one of saidflow-separated components to an x-ray excitation beam to produce anx-ray fluorescence signal therefrom, the x-ray excitation source beingconfigured for exciting atoms of the chemical and of a chemical portionof any bound complex present in any flow-separated component; an x-raydetector configured to detect the x-ray fluorescence signal emitted froma flow-separated component; processing electronics configured todetermine from the x-ray fluorescence signal produced from the excitedatoms present in the chemical and the chemical portion of any boundcomplex present in a separated component whether or not any separatedcomponent comprises a bound complex; and a pump engaged with said flowseparator configured to provide a pressure gradient along said flowseparator.
 2. The apparatus of claim 1 further comprising a diverterconfigured to divert a chosen flow-separated component from a remainingsolution and any other flow-separated component.
 3. The apparatus ofclaim 1 wherein said x-ray excitation beam comprises a polychromaticx-ray excitation beam.
 4. The apparatus of claim 1 wherein said x-rayexcitation beam comprises x-rays having an energy greater than 12 KeV.5. The apparatus of claim 1 wherein said x-ray excitation beam comprisesx-rays having an energy less than 8 KeV.
 6. An apparatus for screeningchemical binding, comprising: a flow separator configured to separate asolution into at least two flow-separated components, the solutioncomprising at least one chemical and at least one target binder; apolychromatic x-ray excitation source configured to expose at least oneof the flow-separated components to a polychromatic x-ray excitationbeam to produce an x-ray fluorescence signal therefrom, thepolychromatic x-ray excitation source being configured for excitingatoms of the chemical and of a chemical portion of any bound complexpresent in any flow-separated component; an x-ray detector configured todetect the x-ray fluorescence signal emitted from a flow-separatedcomponent; and means for determining from the x-ray fluorescence signalproduced from the excited atoms present in the chemical and the chemicalportion of any bound complex present in a separated component whether ornot any separated component comprises a bound complex.
 7. The apparatusof claim 6 wherein said x-ray excitation beam comprises x-rays having anenergy greater than 12 KeV.
 8. The apparatus of claim 6 wherein saidx-ray excitation beam comprises x-rays having an energy less then 8 KeV.9. The apparatus of claim 6 wherein said means for determining whetheror not any separated component comprises a bound complex comprisesprocessing electronics configured to determine from the x-rayfluorescence signal produced from the excited atoms present in thechemical and the chemical portion of any bound complex present in aseparated component whether or not any separated component comprises abound complex.
 10. An apparatus for screening chemical binding,comprising: a flow separator configured to separate a solution ofchemicals and at least one target binder into at least twoflow-separated components; an x-ray excitation source configured toexpose at least one of said flow-separated components to an x-rayexcitation beam comprising x-rays of less than 9 KeV to produce an x-rayfluorescence signal therefrom, the x-ray excitation source beingconfigured for exciting atoms of the chemical and of a chemical portionof any bound complex present in any flow-separated component; an x-raydetector configured to detect the x-ray fluorescence signal emitted froma flow-separated component; and processing electronics configured todetermine from the x-ray fluorescence signal produced from the excitedatoms present in the chemical and the chemical portion of any boundcomplex present in a separated component whether or not any separatedcomponent comprises a bound complex.